β-Catenin, which functions as a downstream transcriptional activator in the Wnt signaling pathway, is a submembrane component of the cadherin-mediated cell-cell adhesion system (Abraham, S. C. et al., Am. J. Pathol. 158:1005-1010, 2001; Abraham, S. C. et al., Am. J. Pathol. 158:1073-1078, 2001). APC (adenomatus polyposis coli) tumor suppressor protein, along with GSK-3β (glycogen synthase kinase-3β), promotes the phosphorylation of the serine/thereonine residues in exon 3 of the β-catenin gene (Abraham, S. C. et al., Am. J. Pathol. 158:1073-1078, 2001). Mutation of the APC gene or the β-catenin gene was found to result in the accumulation of β-catenin protein and the loss of β-catenin regulatory activity (Abraham, S. C. et al., Am. J Pathol. 158:1073-1078, 2001). The majority of β-catenin mutations have been reported at specific GSK-3β phosphorylation sites, i.e., Ser-33, Ser-37, Thr-41, Ser-45, and other residues (Asp-32 and Gly-34) in many human cancers, including endometrial, gastric, ovarian, hepatoblastomas, and colorectal cancers (Saegusa, M. and Okayasu, I. J Pathol. 194:59-67, 2001). In colorectal cancers, various frequencies of the β-catenin mutations have been reported, ranging from 0 to 16% (Nilbert, M. and Rambech, E. Cancer Genet. Cytogenet. 128:43-45, 2001; Mirabelli-Primdahl, L. et al., Cancer Res. 59:3346-51, 1999). Most β-catenin mutations are restricted at some codons in exon 3, and substitution mutations causing amino acid changes predominate in the β-catenin gene (Devereux, T. R. et al., Mol. Carcinog. 31:68-73, 2001; Udatsu, Y. et al., Pediatr Surg. Int. 17:508-512, 2001; Koch, A. et al., Cancer Res. 59:269-273, 1999; de La Coste, A. et al., Proc. Natl. Acad. Sci. USA 95:8847-8851, 1998).
Although it seems easy to detect β-catenin gene mutations using conventional methods, such as PCR-SSCP (single strand conformation polymorphism) and direct sequencing, technical problems associated with the low sensitivity of such β-catenin mutation detection methods have been reported (Abraham, S. C. et al., Am. J. Pathol. 158:1005-1010, 2001). Thus, there has been a need to develop a more reliable and faster mutation detection technique for β-catenin gene which can be used for various cancer studies, e.g., elucidation of the Wnt signaling related mechanism.
Studies have suggested that the high frequency MSI (microsatellite instability-H, MSI-H) colorectal cancer is not linked to APC mutations (Mirabelli-Primdahl, L. et al., Cancer Res. 59:3346-51, 1999), and that β-catenin gene mutations are mainly induced in MSI-H colorectal carcinomas (Mirabelli-Primdahl, L. et al., Cancer Res. 59:3346-51, 1999; Shitoh, K. et al., Genes Chromosomes Cancer 30:32-37, 2001).
Traverso et al. used MSI in the stool as a marker for the diagnosis of proximal colon cancers in stools (Traverso, G. et al., Lancet. 359:403-404, 2002), and several other markers, such as APC, p53, long DNA and K-ras, have been also used for colorectal cancer diagnosis using fecal DNA (Ahlquist, D. A. et al., Gastroenterology 119:1219-1227, 2000; Dong S. M. et al., J. Natl. Cancer Inst. 93:858-865, 2001).
The fact that β-catenin mutations are prone to occur in proximal colon cancers suggests β-catenin mutations might be used to diagnose proximal colon cancer. Accordingly, the present inventors have developed a β-catenin oligonucleotide microchip manufactured by fixing oligonucleotides on the surface of a solid matrix using an automatic microarrayer, the oligonucleotides being designed to detect various mutations at mutational hot spot regions of β-catenin gene. The β-catenin oligonucleotide microchip of the present invention can be used in studies to detect β-catenin mutations and to unravel the signal transduction mechanism and tumorigenesis related to β-catenin gene.